Membrane fusion is a key biological process that enables protein sorting and trafficking, fertilization, and neurotransmitter release. Membrane fusion is mediated by fusogenic proteins that are associated or embedded in the membrane. Much of what is known about cellular membrane fusion has been learned from studying viruses. Enveloped viruses contain a lipid bilayer, acquired from a host cell, which encapsidates the viral genome. Fusion must occur for viruses to release their genome into the cytoplasm membrane. This fusion takes place after a triggering event, such as receptor binding or exposure to low pH conditions. The process of viral fusion is a basic and enduring mystery. Understanding the mechanism of fusion is both of fundamental biological interest, and has significant potential to influence drug development. An inhibitor of membrane fusion in HIV-1 is already on the market. Vesicular stomatitis virus (VSV), a prototype of the Rhabdoviridae family, is an enveloped, non- segmented, negative strand RNA virus. The viral glycoprotein G is the sole protein expressed on the viral surface and is present as ~400 trimeric spikes. G acts as both an attachment factor and a fusion protein. The X-ray crystal structure of VSV G has recently been solved, revealing it as a novel class of fusion proteins, termed class III. VSV G binds to cells through electrostatic interactions. It enters through clathrin-dependent endocytopsis and is trafficked to the endosome pathway. Conformational changes in G are triggered in the endosome, leading to viral membrane fusion. This proposal aims to determine how VSV G, a "class III" viral fusion protein, accomplishes fusion. This will be addressed through a combination of site directed mutagenesis of G, including the construction of fusion incompetent mutants, and a detailed biochemical and biophysical characterization of the properties of those viruses. Specifically, we will determine the effect of mutations in G on virus binding to cells, low pH induced conformational change, and using a novel single viral particle fusion assay, the ability to catalyze hemifusion and fusion pore formation. In addition to this structure-function analysis of G protein, I will test whether multiple G trimers are required for hemifusion and/or fusion pore formation. PUBLIC HEALTH RELEVANCE: Membrane fusion is a key cellular process that is also used by infectious agents. Viruses use fusogenic proteins to accomplish membrane fusion, allowing them to deliver their genetic material to a host cell. Because of this, membrane fusion is an important step to target with antivirals. This study aims to explain how vesicular stomatitis virus (VSV) accomplishes membrane fusion using a novel class of fusion proteins.